CN109477086A - A kind of genomic DNA specificity edit methods and application - Google Patents
A kind of genomic DNA specificity edit methods and application Download PDFInfo
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Abstract
A method of methylation/demethylation state of nucleic acid is adjusted, specifically: a kind of method that one or more methylated bases can be removed by the fixed point in the genome that sgRNA sequence guides in the cell.
Description
The invention belongs to methylation/demethylation state method and its application of technical field of bioengineering more particularly to a kind of specific regulatory genes group DNA.
DNA methylation is one of the important modification in epigenetic regulation, " the 5th kind of base " referred to as in mammalian DNA in addition to tetra- kinds of bases of ATCG.As a kind of covalent modification, DNA methylation plays a significant role in normal differentiation and development and disease occur, while can stablize heredity in the more cell differentiation of higher eucaryote organ, and discovery can be transmitted to the next generation by sperm in zebra fish.Under the influence of cell differentiation, disease and environment, great variety can occur for methylation state of DNA.
Studies have shown that the occurrence and development of DNA methylation and tumour have close contact.The change of methylation state of DNA includes two kinds of hyper-methylation (hypermethylation) and hypomethylation (hypomethylation).In general, the DNA hyper-methylation of gene promoter area has the function of cryptiogene expression, and the expression of hypomethylation then activated gene.The probability for occurring gene mutation in cancerous tumor cell will be well below expection to be shown to the DNA analysis of different tumour cells.And the gene expression inhibition as caused by promoter hyper-methylation in colorectal cancer is detected within the scope of transcript profile, abnormal promoter hyper-methylation has occurred in the known of discovery up to 5% in tumour cell.Therefore it may be speculated that compared with gene mutation, DNA methylation change may play bigger effect during malignant change of cell.
The specificity editor of the nucleic acid editing technique of desired specificities, especially genomic DNA, is always the important technical basis of gene therapy.And go deep into epigenetics research, more and more researches show that the methylation of genome directly takes part in other regulations of transcriptional control and genome, and the methylation level of its promoter of gene and enhancer region in expression active state is often in relatively low state.Therefore, a kind of nucleotide editing technique for capableing of specific demethylation has very important meaning for the transcriptional activation for the gene that sinks.
Currently, specific site and the demethylation of compartmentalization all have been reported.For example the genome remodeling of reproduction cell is usually associated with large-scale demethylation.In addition, 5mC can be oxidized to 5hmC and further achieve the purpose that demethylation by BER or NER approach by certain enzymes (such as Tet).Xu Guoliang et al. has reported and has applied the patent of the method by the reagents demethylation such as Tet dioxygenase and thymidine DNA glycosylase in 2015, but this method can not accurately edit some site, become the important bottleneck applied to gene therapy or experimental technique tool.
Certain members of Apobec protein family have the ability that 5mC deamination is changed into T in single-stranded DNA.By such characteristic and the pinpoint ability of CRISPR protein family, developing a kind of the system of specific site methylation can become possibility in accurate edits genome.
Summary of the invention
To solve the above-mentioned problems, the present invention provides a kind of method accurately edited to nucleic acid molecules, described method includes following steps:
(1) recombinant vector of encoding fusion protein (A) and single-stranded guiding RNA (sgRNA) (B) are obtained, the fusion protein (A) includes the Cas9 family or Cpf1 protein structure domain that the Apobec family protein structural domain positioned at nitrogen end and the nuclease positioned at carbon teminal inactivate, single-stranded guiding RNA and the target editing area of target nucleic acid molecules have complementary region, wherein the cytidylic acid comprising at least one methylation in the target editing area of target nucleic acid molecules；
(2) recombinant vector of the encoding fusion protein (A) and single-stranded guiding RNA (sgRNA) (B) that obtain step (1) is contacted with target nucleic acid molecules.
Recombinant vector in above-mentioned steps, it can be the recombinant vector that two carriers are separately encoded fusion protein (A) and single-stranded guiding RNA (sgRNA) (B), be also possible to a recombinant vector encoding fusion protein (A) and the single-stranded recombinant vector for being oriented to RNA (sgRNA) (B) simultaneously.
In a preferred case, people Apobec3A or Apobec3H, or the albumen with deamination activity with people Apobec3A or Apobec3H with 95% or more homology are selected from positioned at the Apobec family protein of nitrogen end in fusion protein.Preferred Apobec albumen is Apobec 3H or Apobec 3A.
Under another preferable case, be in wild type Cas9 albumen positioned at the Cas9 family protein that the nuclease of carbon teminal inactivates in fusion protein by the aspartic acid in the 10th and 840 sites and Histidine mutagenesis be alanine and alanine, or positioned at carbon teminal the Cpf1 family that inactivates of nuclease but by by the Aspartic acid mutations in the 908th site to be alanine in wild type Cpf1 albumen.
To make two protein structure domains of fusion protein that there is the flexibility on better space structure, the connector being made of 3-14 motif can be increased between two structural domains of fusion protein.The motif is selected from (GGS).Connector is longer, and the space flexibility of albumen is higher, and editable target area is bigger.
For convenience of the expression and purification of fusion protein, it may also include purification tag sequence.Usually used purification tag is 6xHis.
In a more preferred case, the fusion protein is selected from any sequence of SEQ ID No 201-207.
The present invention also provides the gene order for encoding above-mentioned fusion protein sequence, affiliated gene order is selected from shown in SEQ ID No 301-307 under preferable case.
The present invention also provides the recombinant vector comprising any of the above-described gene order, it can be prokaryotic expression carrier or carrier for expression of eukaryon, including but not limited to plasmid vector, viral vectors etc. that the carrier, which regards follow-up test mesh,.
Another aspect of the present invention provides single-stranded guiding RNA molecule.In a preferred case, the single-stranded guiding RNA length is 60-80bp.Under another preferable case, the single-stranded guiding RNA is 18-25bp, preferably 20bp with the complementary section length of target nucleic acid molecules.
A kind of method of external editor's target nucleic acid molecules, includes the following steps:
(1) fusion protein (A) and single-stranded guiding RNA (sgRNA) (B) are obtained, the fusion protein (A) includes the Cas9 family protein structural domain that the Apobec family protein structural domain positioned at nitrogen end and the nuclease positioned at carbon teminal inactivate, single-stranded guiding RNA and the target editing area of target nucleic acid molecules have complementary region, wherein the cytidylic acid comprising at least one methylation in the target editing area of target nucleic acid molecules；
(2) target nucleic acid molecules are contacted with fusion protein (A) and single-stranded guiding RNA (sgRNA) (B)；
(3) a effective amount of TDG is added after terminating reaction in high temperature, 42 DEG C reaction 6-8 hours；
(4) be added a effective amount of EDTA, formamide and NaOH collective effect, 90-95 DEG C reaction 5-10 minutes.
The present invention also provides application of the method for editor's target nucleic acid molecules in specific regulatory genes group DNA methylation/demethylation state.
The method of editor's target nucleic acid molecules in the present invention, target nucleic acid molecules contain the cytidylic acid of at least one methylation, the diseases such as the cytidylic acid of the methylation and cancer, genetic disorder genetic disease, development mistake are related, the method of editor's target nucleic acid molecules can be used for methylating with cytidylic acid the treatment of related disease, and the disease includes but is not limited to disease relevant to cell prosoplasia.
Beneficial effects of the present invention:
Apobec albumen with deamination activity is guided to the methylated cytosine position of target nucleic acid molecules, is modified methylated cytosine by the guiding function of sgRNA and the specific bond function of mutant Cas9 or Cpf1 by the present invention.Again by internal DNA repair mechanism, methylated cytosine is removed, the specificity of target nucleic acid molecules is edited to realize.Gene editing method of the invention, specificity is high, to target site upstream and downstream sequence no dependence, thus has general applicability.And gene editing method of the invention only edits target, will not generate undershooting-effect, and insertion or deletion mutation will not be introduced in editing process, has lower toxic side effect.
Fig. 1, fusion protein extracellularly edit schematic diagram
Fig. 2, fusion protein edit schematic diagram into the cell
Fig. 3, the activity intensity and range test of several fusion proteins in vitro experiment
Fig. 4 edits influence of the previous bit base of target spot to editorial efficiency
Fig. 5, edited result in two groups of HEK293 cell lines
Fig. 6, two kinds of fusion proteins the same area edited result in PC3 cell line
Cas9 Cpf1 albumen is a kind of double-stranded DNA nuclease, can be integrated under single-stranded guiding RNA (small guide RNA, sgRNA) effect in targeting sequence and cut to double-stranded DNA.The Cas9 albumen of nuclease inactivation retains the activity in conjunction with targeting sequence, but does not cut to target site.The present invention by Cas9 the Cpf1 albumen that inactivates nuclease with the Apobec protein fusion of deamination activity, Apobec albumen is directed to the targeting sequence area of target nucleic acid molecules by the Cas9 albumen or Cpf1 albumen of mutation, deamination is carried out to the methylated cytosine in targeting sequence area, target Met-C will become T under deamination, with G one protrusion of unpaired formation on complementary strand.By high temperature, (main function is by high temperature deactivation fusion protein, the temperature generallyd use is 90-95 degree) it terminates and is added a effective amount of TDG after reaction, the T base for having no idea normally to match can be removed, to form a missing in the editor target position of substrate.DsDNA can become ssDNA again again and be broken in base deletion site under the collective effect of a effective amount of EDTA, Formamide (formamide) and NaOH later.
Based on above-mentioned experiment, it is found by the applicant that fusion protein Apobec-dCas9 or Apobec-dCpf1 can be realized the fixed point editor to the methylated cytosine site in targeting sequence area, upstream and downstream sequence of this fixed point editor independent of methylated cytosine site, with universality, nor undershooting-effect can occur, other insertions or deletion mutation will not be introduced, therefore without other toxic side effects.
It will be further described below by specific embodiment.It should be appreciated, however, that the specific embodiments are only for explaining the present invention, it is not intended to limit the scope of protection of the present invention.The instrument use herein arrived, equipment, reagent, method etc. are instrument commonly used in the art, equipment, reagent and method as not specified.
The expression of one recombinant protein of embodiment and purifying
Invitrogen company is entrusted to synthesize 6His-NLS-Apobec3H-linker (GGS-GGS-GGS)-dCas9 (Asp10Ala/His840Ala), 6His-NLS-Apobec3H-linker (GGS-GGS-GGS-GGS-GGS-GGS-GGS), 6His-NLS-Apobec3H-linker (GGS-GGS-GGS-GGS-GGS-GGS-GGS-GGS-GGS-GGS-GGS-GGS-GGS-GGS)-dCas9 (Asp10Ala/His840Ala), 6His-NLS-Apobe C3A-linker (GGS-GGS-GGS)-dCas9 (Asp10Ala/His840Ala)-dCas9 (Asp10Ala/His840Ala), 6His-NLS-Apobec3A-linker (GGS-GGS-GGS-GGS-GGS-GGS-GGS)-dCas9 (Asp10Ala/His840Ala), 6His-NLS-Apobec3A-linker
(GGS-GGS-GGS-GGS-GGS-GGS-GGS-GGS-GGS-GGS-GGS-GGS-GGS-GGS)-dCas9 (Asp10Ala/His840Ala), 6His-NLS-Apobec3H-linker (GGS-GGS-GGS-GGS-GGS-GGS-GGS)-dCpf1 (Asp908Ala) gene order, respectively Seq ID No 301, No 302, No 303, No 304, No 305, No 306 and No 307, and I restriction enzyme site of Nco is introduced at the end genetic fragment 5', the end 3' introduces III restriction enzyme site of Hind.By the genetic fragment of synthesis and pET28a (+) carrier respectively through Nco I and III double digestion of Hind, T4DNA ligase connects genetic fragment and carrier segments, routine transformation DH5 α competent cell (TIANGEN Biotech (Beijing) Co., Ltd.), according to kanamycin resistance screening positive colony, plasmid is extracted.Recombinant plasmid is identified through Nco I and III double digestion of Hind and agarose gel electrophoresis, Invitrogen company is entrusted to carry out sequencing to recombinant plasmid simultaneously, sequencing result is analyzed using BioEdit software, it is as a result identical as implementation sequence, illustrate that recombinant bacterium constructs successfully.
The positive colony plasmid of acquisition is transformed into E.Coli.BL21 (DE3) competent cell (TIANGEN Biotech (Beijing) Co., Ltd.), received containing 100ug/ml card mycin LB culture medium in 37 degree of overnight incubations, be transferred to 37 degree of cultures in the same LB culture medium of 1L later and arrive OD=0.6 or so.Culture medium is cooled to 4 degree later, the IPTG inducing expression of 0.5mM is added about 16 hours.Thallus is collected by centrifugation by 4000g, is suspended again with lysis buffer (50mM Tris pH=7.0,1M NaCl, 20%glycerol, 10mM TCEP).Thallus cracks (6W is exported 8 minutes, is opened within 20 seconds 20 seconds and is closed) by ultrasonic method, and supernatant is centrifugated by 25000g.Supernatant and Nickel resin (ThermoFisher) are set 1 hour altogether under 4 degree, then passes through gravity column and cracks buffer solution with 40ml and wash.Recombinant protein is eluted with the cracking buffer solution containing 285mM, is diluted to the NaCl of 0.1M and is concentrated into suitable concentration with centrifuge tube.The quality and concentration of recombinant protein are determined by SDS-Page.
Recombinant protein sequence is Seq ID No 201-207.
Two sgRNA of embodiment is transcribed in vitro
According to the 34 of quasi- test dsDNA substrate sequences (Seq ID No 39-54 and its complementary strand 55-70,71-85 and its complementary strand 86-100,101-104 and its complementary strand 105-108) and provide sgRNA universal sequence pFYF320 carrier sequence, separately design sgRNA forward primer (Seq ID No 2-17,18-34,35-38) and reverse primer (Seq ID No 1).SgRNA leads to TranscriptAid T7High Yield Transcription Kit (ThermoFisher Scientific) by the linear DNA fragment containing T7 promoter and obtains, template DNA is removed with DpnI, then it is purified using MEGAclear Kit (ThermoFisher Scientific), quality is detected by UV absorption.
The preparation of three substrate of embodiment
Commission Invitrogen company is respectively synthesized the positive and negative oligonucleotide chain-ordering of substrate sequence, wherein 5 ' end flag F AM fluorescent markers of positive strand sequence.The single-stranded oligonucleotide chain of 2OD is dissolved in 500ul water respectively, the positive anti-chain solution of equivalent is mixed, stands 5 minutes, is obtained double stranded substrate (dsDNA).
For dCas9 fusion protein demethylation range test 15, Serial No. SEQ ID No.39-54.
For dCpf1 fusion protein demethylation range test 15, Serial No. SEQ ID No.71-85.
For testing the previous bit base of target spot 4 to activity influence, Serial No. SEQ ID No.101-104.
Example IV external activity test
The recombination fusion protein that embodiment one is obtained is mixed according to the sgRNA that the ratio of molar ratio 1:1 is obtained with embodiment two respectively, is placed 5 minutes at room temperature.Corresponding dsDNA substrate is added to final concentration 125nM, is reacted 2 hours at 37 DEG C.It is obtained 37 degree of TDG (NEB) reactions 1 hour of 1 unit of addition after the dsDNA reacted using EconoSpin micro spin column (Epoch Life Science) purifying.10ul Formamide, 1ul 0.5M EDTA and 0.5ul 5M NaOH are added after reaction, is reacted 5 minutes at 95 DEG C.Product separates in 10% TBE- urea glue.
It include target Met-C in target dna chain, and 3 ' ends are marked by fluorophor FAM.Under recombinant protein effect, Met-C is changed into T to match with the G of complementary strand.Under the action of TDG, the T that can not be matched can be removed, and leave base deletion site.Double-strand will become single-stranded and further be cut off in base deletion site under the action of Formamide and NaOH, to form the short chain marked by fluorophor FAM.Length chain marker DNA is separated in urea glue.If running cementing fruit long short strips occurs, illustrate that recombinant protein is active.
The preparation of five pyrosequencing dsDNA substrate of embodiment
Commission Invitrogen company is respectively synthesized the positive and negative oligonucleotide chain-ordering of substrate sequence, wherein 5 ' end flag F AM fluorescent markers of positive strand sequence.The single-stranded oligonucleotide chain of 2OD is dissolved in 500ul water respectively, the positive anti-chain solution of equivalent is mixed, stands 5 minutes, is obtained double stranded substrate (dsDNA).The recombination fusion protein that embodiment one is obtained is mixed according to the sgRNA that the ratio of molar ratio 1:1 is obtained with embodiment two respectively, is placed 5 minutes at room temperature.Corresponding dsDNA substrate is added to final concentration 125nM, is reacted 2 hours at 37 DEG C.Transfer to Hua Da gene by carrying out pyrosequencing after sulphite processing and its primer amplification designed after obtaining the dsDNA reacted using EconoSpin micro spin column (Epoch Life Science) purifying.
Determination of activity in embodiment hexasomic
(1) cell culture
HEK293 cell line or PC3 cell line maintain under 37 DEG C of 5% carbon dioxide environment in Dulbecco ' s Modified Eagle ' s Medium plus.
(2) PX330 recombinant protein expression vector establishment
Invitrogen company is entrusted to synthesize 6His-NLS-Apobec3H-linker (GGS-GGS-GGS)-dCas9 (Asp10Ala/His840Ala), 6His-NLS-Apobec3H-linker (GGS-GGS-GGS-GGS-GGS-GGS-GGS), 6His-NLS-Apobec3H-linker (GGS-GGS-GGS-GGS-GGS-GGS-GGS-GGS-GGS-GGS-GGS-GGS-GGS-GGS)-dCas9 (Asp10Ala/His840Ala), 6His-NLS-Apobe C3A-linker (GGS-GGS-GGS)-dCas9 (Asp10Ala/His840Ala)-dCas9 (Asp10Ala/His840Ala), 6His-NLS-Apobec3A-linker (GGS-GGS-GGS-GGS-GGS-GGS-GGS)-dCas9 (Asp10Ala/His840Ala), 6His-NLS-Apobec3A-linker (GGS-GGS-GGS-GGS-GGS-GGS-GGS-GGS-GGS-GGS-GGS-GGS-GGS-GGS)-dCas9 (Asp10Ala/His 840Ala), 6His-NLS-Apobec3H-linker (GGS-GGS-GGS-GGS-GGS-GGS-GGS)-dCpf1 (Asp908Ala) gene order, respectively Seq ID No 301, No 302, No 303, No 304, No 305, No 306 and No 307, and BamHI restriction enzyme site is introduced at the end genetic fragment 5', the end 3' introduces AgeI restriction enzyme site.By the genetic fragment of synthesis and pX330 carrier (Addgene) respectively through BamHI and AgeI double digestion, T4DNA ligase connects genetic fragment and carrier segments.It is correct through sequencing confirmation construction of recombinant vector.
Corresponding PCR product (being obtained by forward primer 121,123,125,127,129 and reverse primer 1,122,124,126,128,130 by PCR) is inserted by corresponding five groups of sgRNA carriers tested into the cell through MluI and SpeI double digestion.
(3) it transfects
A. the day before transfection, HEK293 cell or PC3 cell are in the inoculation of medium for not including antibiotic, until the convergence degree of cell is 30~50% when transfection.
B. transfection sample is prepared:
The pX330 recombinant vector and 1.5 μ l lipofectamine Lipofectamine for being respectively 20 μM by 1 μ l concentrationTM2000 (Invitrogen) are diluted in 0.05ml Opti-MEM (Invitogen), mix gently, and are incubated for 5 minutes.Control group is using the blank pX330 carrier for not cloning any foreign gene.
Take diluted pX330 recombinant vector and LipofectamineTM2000 (Invitrogen) are incubated at room temperature 20 minutes, form (Invitrogen) compound of recombinant vector-Lipofectamine 2000 and empty vectors-Lipofectamine 2000
(Invitrogen) compound.Incubation time is no more than 30 minutes, and longer incubation time may reduce activity.
Carrier-LipofectamineTM2000 compound is added in hole that each includes cell and culture medium, by the culture plate mixing that lightly rocks back and forth, at 37 DEG C, CO2Incubator is incubated for 72 hours.
It is harvested behind cell 3 days after transfection, genomic DNA is purified by Agencourt DNAdvance Genomic DNA Isolation Kit (Beckman Coulter) and obtained.Sample preparation is carried out by the method for embodiment five, gained sample transfers to Shenzhen Hua Da company to carry out pyrosequencing.
The measurement of seven demethylation action site range of embodiment
According to embodiment two, inventor has synthesized ssDNA that 30 (15 are directed to the fusion protein of dCas9, and 15 are directed to the fusion protein of dCpf1) length are 59 bases as reaction substrate, their complementary ssDNA and corresponding sgRNA primer.Wherein 5 ' the ends of reaction substrate ssDNA are modified by fluorophor FAM, and there are the C (Met-C) of a methylation modification, that is, the target edited in centre.SsDNA is complementary chain and is formed after dsDNA substrate, and the region Cas9 of recombinant protein can be integrated to the corresponding region among dsDNA under corresponding sgRNA guidance, and by the base unwinding of about 20, the region, that is, a single stranded zone is formed among dsDNA.Target Met-C just in this region, and is named as substrate 4-20 according to its distance (4-20 base) to 5 ' end double stranded regions.After recombinant protein is in conjunction with different sgRNA again with corresponding dsDNA substrate-function after a certain period of time, a part of target Met-C will become T under deamination, with G one protrusion of unpaired formation on complementary strand.The TDG that addition 1Unit after reaction is terminated by high temperature, can remove the T base for having no idea normally to match, to form a missing in the editor target position of substrate.DsDNA can become ssDNA again again and be broken in base deletion site under the collective effect of EDTA (0.5ul concentration is 0.5M), Formamide (10ul) and NaOH (1ul 5M) later.Since 5 ' the short chain ssDNA in end of fracture and the ssDNA substrate that is not applied have specific FAM fluorophor to mark at 5 ' ends, the relative scale of the two can be accurately calculated, and infer that Met-C is become in the site functioning efficiency of T by recombinant protein with this.
Such as Fig. 3, pass through the experimental result to 15 different substrates, it can be seen that the dCas9 fusion protein for being (GGS) 3 for linker, with 5 of single-stranded regions after the double-strand unwinding of target area ' end first base distance can be changed into T for the Met-C in 7-10 base, and surpass then will not of going beyond the scope；And for linker it is the fusion protein of (GGS) 7 and (GGS) 14, the distance of this edit interval is 6-11 base and 5-13 base.This range can slightly broaden because of the length of linker.This range will be the important evidence of experimental design and the following gene therapy design sgRNA after us.
Can also see that the activity of A3H from result more by force than A3A outline.
From the results, it was seen that the albumen that the dCpf1 that linker length is (GGS) 7 is merged also has similar activity, the distance for acting on section is 7-12 base.
Control group experiment, the T of positive synthesis, negative uses mistake sgRNA and the not Cas-9 or Cpf1 of sgRNA.
Control experiment is mainly for two problems of proof: first, our method is feasible.Selection wherein some be clearly visible the group that short chain DNA is formed, we synthesize same ssDNA substrate, but intermediate Met-C are become T, that is, artificially complete the function of recombinant protein.Later using same operation.As a result the appearance of short chain DNA is equally seen.Prove that the short chain DNA in experimental result is strictly to be acted on generating on target dna by recombinant protein；Second, by allowing recombinant protein not combine or being further continued for next experiment flow in conjunction with unpaired sgRNA, there is no short chain DNAs to generate, it was demonstrated that such editor is orientation.
The influence of eight action site upstream and downstream base of embodiment
Use recombinant protein (linker GGS*7, Apobec albumen is A3H) effect editor's previous bit base of target spot to the research object of the activity influence of demethylation.
Research based on forefathers to Apobec protein family, its activity of the base-pair of the editor previous position of target spot have direct influence.We have chosen the substrate that Met-C is located at No. 7 positions, and changing previous bit base respectively is A, T, C and G.Such as Fig. 4, test result is shown, the sequence of previous bit base does not influence editorial efficiency substantially, it was demonstrated that the versatility of this technology.
The efficiency of the intracellular demethylation of embodiment nine
When we have demonstrated that the recombinant protein after the extracellular ability for having preferably and Met-C being become to T, it is desirable that further verifying in the cell such activity whether still remain, the T after activity intensity and reaction whether can be by the DNA repair mechanism reparation of cell itself at common C, to have the function that pinpoint demethylation.Applicant devises three groups and tests into the cell, and the promoter region that we have chosen three different genes respectively carries out demethylation test.
First intracellular editor's target is two methylation C in 17741472 and 17741474 sites of No. 11 chromosome, positioned at the promoter region of gene M YOD1 in HEK293 cell line.Such as Fig. 5, this is the experiment proves that this system can accurately edit that of our selections in the close methylation modification in two positions.
Second editor's target is a methylation C in No. 6 31138558 sites of chromosome, positioned at the promoter region of gene POUF1 in HEK293 cell line.Such as Fig. 5, this experiment also obtains ideal edit effect.
It is a methylation C in 113875226 sites of No. 2 chromosome, positioned at the promoter region of gene IL1RN in PC3 cell line that third, which edits target,.Such as Fig. 6, this system designs one or two sites in two methylation sites that can be adjacent by reasonable sgRNA.
Recombinant vector is constructed using method described in embodiment six respectively and is transfected to cell, then edited result is assessed by pyrosequencing.
There is the ratio of indel (insertion and deletion) after editing into the cell in embodiment ten
Based on the sequencing result of above-mentioned experiment, we have counted the case where base occurred near target site in the whole process is inserted into (insertion) and missing (deletion) simultaneously.It looks around from sequencing result the phenomenon that being not inserted into and lacking base appearance.
Shown in the table specific as follows of nucleic acid sequence used in embodiment.
Protein structure domain sequence is as follows:
Seq ID NO201:
Seq ID NO202:
Seq ID NO203:
Seq ID NO204:
Seq ID NO205:
Seq ID NO206:
Seq ID NO207:
Seq ID NO301:
Seq ID NO302:
Seq ID NO303:
Seq ID NO304:
Seq ID NO305:
Seq ID NO306:
Seq ID NO307:
Claims (15)
- A method of editor's target nucleic acid molecules, described method includes following steps:(1) recombinant vector of encoding fusion protein (A) and single-stranded guiding RNA (sgRNA) (B) are obtained, the fusion protein (A) includes the Cas9 family or Cpf1 family protein structural domain that the Apobec family protein structural domain positioned at nitrogen end and the nuclease positioned at carbon teminal inactivate, single-stranded guiding RNA and the target editing area of target nucleic acid molecules have complementary region, wherein the cytidylic acid comprising at least one methylation in the target editing area of target nucleic acid molecules；(2) recombinant vector of the encoding fusion protein (A) and single-stranded guiding RNA (sgRNA) (B) that obtain step (1) is contacted with target nucleic acid molecules.
- The method of editor's target nucleic acid molecules according to claim 1, it is characterized in that being selected from people Apobec3A or Apobec3H, or the albumen with deamination activity with people Apobec3A or Apobec3H with 95% or more homology positioned at the Apobec family protein of nitrogen end in fusion protein.
- The method of editor's target nucleic acid molecules according to claim 1, it is characterized in that aspartic acid and Histidine mutagenesis of the protein sequence of the Cas9 albumen inactivated in fusion protein positioned at the nuclease of carbon teminal in the 10th and 840 sites are the mutant nucleotide sequence of alanine and alanine, or positioned at carbon teminal Aspartic acid mutations of the protein sequence of Cpf1 albumen that loses of nuclease in the 908th site be alanine mutant nucleotide sequence.
- The method of editor's target nucleic acid molecules according to claim 1, it is characterised in that be the connector of 3-14 motif composition between two structural domains of fusion protein.
- The method of editor's target nucleic acid molecules according to claim 4, it is characterised in that the motif is selected from (GGS).
- The method of editor's target nucleic acid molecules according to claim 1, it is characterised in that the fusion protein further includes purification tag sequence.
- The method of editor's target nucleic acid molecules according to claim 1-6, it is characterised in that the fusion protein is selected from any sequence of SEQ ID No 201-207.
- Encode the gene order of protein sequence described in claim 7.
- Recombinant vector comprising any gene order of claim 8, the carrier are selected from plasmid vector or viral vectors.
- The method of editor's target nucleic acid molecules according to claim 1, it is characterised in that the single-stranded guiding RNA length is 60-80bp.
- The method of editor's target nucleic acid molecules according to claim 1, it is characterised in that the single-stranded guiding RNA is 18-25bp with the complementary section length of target nucleic acid molecules.
- A kind of method of external editor's target nucleic acid molecules, described method includes following steps:(1) fusion protein (A) and single-stranded guiding RNA (sgRNA) (B) or encoding fusion protein (A) and single-stranded are obtained It is oriented to the recombinant vector of RNA (sgRNA) (B), the fusion protein (A) includes the Cas9 family protein structural domain that the Apobec family protein structural domain positioned at nitrogen end and the nuclease positioned at carbon teminal inactivate, single-stranded guiding RNA and the target editing area of target nucleic acid molecules have complementary region, wherein the cytidylic acid comprising at least one methylation in the target editing area of target nucleic acid molecules；(2) target nucleic acid molecules are contacted with fusion protein (A) and single-stranded guiding RNA (sgRNA) (B)；(3) a effective amount of TDG is added after terminating reaction in high temperature, 42 DEG C reaction 6-8 hours；(4) be added a effective amount of EDTA, formamide and NaOH collective effect, 90-95 DEG C reaction 5-10 minutes.
- According to claim 1 or the method for editor's target nucleic acid molecules described in 12, it is characterised in that the diseases such as the cytidylic acid of the methylation and cancer, genetic disorder genetic disease, development mistake are related.
- Application of the method for the editor's target nucleic acid molecules of claim 1 or 12 in specific regulatory genes group DNA methylation/demethylation state.
- Application of the method for editor's target nucleic acid molecules described in claim 1 in the diseases such as treating cancer, genetic disorder genetic disease, development mistake.
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| PCT/CN2017/088281 WO2018010516A1 (en) | 2016-07-13 | 2017-06-14 | Method for specifically editing genomic dna and application thereof |
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| WO2019041296A1 (en) * | 2017-09-01 | 2019-03-07 | 上海科技大学 | Base editing system and method |
| CN109021111B (en) * | 2018-02-23 | 2021-12-07 | 上海科技大学 | Gene base editor |
| CN111788232A (en) * | 2018-02-23 | 2020-10-16 | 上海科技大学 | Fusion proteins for base editing |
| CN108753823B (en) * | 2018-06-20 | 2022-09-23 | 李广磊 | Method for realizing gene knockout by using base editing technology and application thereof |
| CN111165342A (en) * | 2020-01-19 | 2020-05-19 | 安徽省农业科学院水稻研究所 | Breeding method of indica-type rice restorer line |
| CN114540325B (en) * | 2022-01-17 | 2022-12-09 | 广州医科大学 | Method for targeted DNA demethylation, fusion protein and application thereof |
| WO2023155901A1 (en) * | 2022-02-17 | 2023-08-24 | Correctsequence Therapeutics | Mutant cytidine deaminases with improved editing precision |
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| WO2015089406A1 (en) * | 2013-12-12 | 2015-06-18 | President And Fellows Of Harvard College | Cas variants for gene editing |
| WO2015133554A1 (en) * | 2014-03-05 | 2015-09-11 | 国立大学法人神戸大学 | Genomic sequence modification method for specifically converting nucleic acid bases of targeted dna sequence, and molecular complex for use in same |
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| WO2016025131A1 (en) * | 2014-08-13 | 2016-02-18 | E. I. Du Pont De Nemours And Company | Genetic targeting in non-conventional yeast using an rna-guided endonuclease |
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| KR102210322B1 (en) * | 2013-03-15 | 2021-02-01 | 더 제너럴 하스피탈 코포레이션 | Using rna-guided foki nucleases (rfns) to increase specificity for rna-guided genome editing |
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2017
- 2017-06-14 CN CN201780043459.1A patent/CN109477086A/en active Pending
- 2017-06-14 US US16/317,524 patent/US20230151341A1/en not_active Abandoned
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| WO2015089406A1 (en) * | 2013-12-12 | 2015-06-18 | President And Fellows Of Harvard College | Cas variants for gene editing |
| WO2015133554A1 (en) * | 2014-03-05 | 2015-09-11 | 国立大学法人神戸大学 | Genomic sequence modification method for specifically converting nucleic acid bases of targeted dna sequence, and molecular complex for use in same |
| WO2016025131A1 (en) * | 2014-08-13 | 2016-02-18 | E. I. Du Pont De Nemours And Company | Genetic targeting in non-conventional yeast using an rna-guided endonuclease |
| CN105112446A (en) * | 2015-06-25 | 2015-12-02 | 中国医学科学院基础医学研究所 | Method for high-efficiency establishment of genetically modified animal model through haploid stem cells |
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| WO2018010516A1 (en) | 2018-01-18 |
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